Numerous reports have indicated that opioid peptides such as methionine-enkephalin (met-enkephalin) have profound effects on the immune system. Previous studies from our and other laboratories have shown that this modulation of immune responses is not restricted to the intact enkephalin molecule; peptides consisting of the first two (Tyr-Gly) and three amino acids (Tyr-Gly-Gly) from the intact pentapeptide are bioactive. However, the mechanisms underlying these observations have yet to be fully elucidated. The long term goal of this project is to gain insight into two the enkephalins and certain hydrolysis fragments derived from these molecules are able to positively or negatively modulate immune responses. This understanding will provide a basis for manipulating these effects in stress, disease and other maladies. Previous and ongoing studies in this laboratory support our central hypothesis: immunomodulating by the enkephalins is due in part, if not entirely, to two peptide fragments : Tyr-Gly-Gly (YGG) and Tyr-Gly (YG). Other hypotheses to be tested in this study are: a) when met enkephalin binds to classical opiate receptors on immune cells, membrane ectoenzymes cleave the molecule into various fragments including inactive forms, and b) YGG and YG bind to and mediate their effects on immune cells through nonclassical opiate receptors. An animal model developed in this lab using Mycobacterium tuberculosis-immunized IAF/HA-HO hairless guinea pigs challenged with purified protein derivative will be used to determine if protease inhibitors either block or enhance modulation of delayed-type hypersensitivity. Responses by met-enkephalin, YGG and YG. In vitro assays using mouse splenocytes (in serum-free medium) stimulated with concanavalin A to induce IL-2, interferon-gamma and IL-4 (assayed via ELISA) will address: 1) whether antagonists to classical opioid receptors (mu, delta and kappa) inhibit the immunomodulatory activity of met-enkephalin and its peptide fragment s; 2) if modulation of cytokine production by met-enkephalin, YGG and YG correlates with cell proliferation (BrdU ELISA) and cytokine mRNA levels (assayed via PCR); 3) if cell lines (stimulated with phorbol 12-myristate 13-acetate) and purified CD4+ cells (stimulated with anti-CD3) are receptive to immunomodulation by the bioactive peptides (to help characterize the nature of the responding cell); 4) whether cells used in our in vitro studies degrade met-enkephalin into YGG and YG (using HPLC to follow production of peptide fragments); 5) whether enkephalin-degrading enzymes are released in soluble form; and 6) if any observed enzymatic activity is blocked by specific protease inhibitors. These studies will form the basis for future investigations into the immunoregulatory effects of these endogenous peptides during disease processes and other conditions.